Method for preparing long aliphatic chain diacid derivative and application of long aliphatic chain diacid derivative

Abstract

The invention provides a method for preparing a long aliphatic chain diacid derivative and application of the long aliphatic chain diacid derivative, and the method comprises the following steps: carrying out acylating chlorination reaction on long aliphatic chain diacid with a structure as shown in a formula (A), and reacting with tert-butyl alcohol to obtain long aliphatic chain diacid-tert-butyl ester; the preparation method comprises the following steps: sequentially carrying out esterification reaction, nucleophilic addition amidation reaction with a compound as shown in a formula (B), hydrogenation reaction, nucleophilic addition amidation reaction with a compound as shown in a formula (E), esterification reaction with N-hydroxysuccinimide and nucleophilic addition amidation reaction with a compound as shown in a formula (H) on long aliphatic chain diacid tert-butyl ester; and carrying out esterification reaction with N-hydroxysuccinimide, and finally carrying out deprotection reaction to obtain the compound as shown in the formula (K). The method has the advantages of simplicity and convenience in operation, low content of related impurities in the product, easiness in purification of an intermediate, high yield, simplicity in purification of the product, commercialization and the like, and a new idea is provided for preparation of long-acting insulin.

Description

[0001] Priority Information

[0002] This invention claims the priority and benefits of the patent application 202311593593.2 filed on November 27, 2023, and incorporates the entire text herein by reference. Technical Field

[0003] This invention relates to the field of biopharmaceuticals, and particularly to a method for preparing long-chain fatty diacid derivatives and their applications. Background Art

[0004] Long-acting insulin is a type of insulin classified by its action time, with a relatively long action time (24 hours or more), and is used for the treatment of type 1 and type 2 diabetes. Usually, only one injection is required per day, and there is no obvious action peak in the body, mainly providing basal insulin. Currently, the mainstream long-acting insulins are insulin glargine, insulin detemir, and insulin degludec, and the latter two are obtained by using long-chain fatty acid modification technology.

[0005] Although long-chain fatty diacid derivatives can be used in the research and development of long-acting insulin drugs through general protein modification techniques, in the prior art, in the preparation process of long-chain fatty diacid derivatives, the chemical reagent cost is high, the selectivity is poor, there are many by-products and it is difficult to purify, making it difficult to achieve industrial scale-up.

[0006] Therefore, there is still a need for further development and improvement in the preparation method of long-chain fatty diacid derivatives. Summary of the Invention

[0007] This invention aims to solve at least one of the technical problems in the related art to some extent. For this purpose, this invention provides a method for preparing monoter-butyl long-chain fatty diacid, which is a key intermediate for preparing long-chain fatty diacid derivatives and long-acting insulin, providing a new idea for the preparation of long-chain fatty diacid derivatives and long-acting insulin.

[0008] For this purpose, the first aspect of this invention provides a method for preparing monoter-butyl long-chain fatty diacid, including the following steps:

[0009] S1: Subject the long-chain fatty diacid to an acyl chlorination reaction to obtain long-chain fatty diacyl chloride;

[0010] S2: Contact the long-chain fatty diacyl chloride with tert-butanol and organic base reagent a to obtain monoter-butyl long-chain fatty diacid;

[0011] Wherein, the long-chain fatty diacid has the structure shown in formula (A);

[0012]

[0013] X is an integer from 6 to 32.

[0014] The method for preparing monoter-butyl long-chain fatty diacid provided by the present invention has the advantages of simple operation, low content of product-related impurities, easy purification of intermediates, high yield, simple purification of products, and commercialization, and can be used for subsequent preparation of long-chain fatty diacid derivatives and long-acting insulin.

[0015] According to an embodiment of the present invention, step S1 further includes subjecting the long-chain fatty diacid to an acyl chlorination reaction with an acyl chlorination reagent to obtain long-chain fatty diacyl chloride.

[0016] According to an embodiment of the present invention, the molar amount of the acyl chlorination reagent is equivalent to 3 to 16 times the molar amount of the long-chain fatty diacid.

[0017] According to an embodiment of the present invention, the acyl chlorination reagent includes at least one selected from thionyl chloride, phosphorus pentachloride, phosphorus trichloride, phosphorus oxychloride, and oxalyl chloride.

[0018] According to an embodiment of the present invention, the acyl chlorination reaction is carried out at a temperature of 70 °C to 120 °C for 3 to 24 hours or in the presence of a catalyst at a temperature of 15 °C to 30 °C.

[0019] According to an embodiment of the present invention, the catalyst is N,N-dimethylformamide.

[0020] According to an embodiment of the present invention, the organic base reagent a in step S2 includes at least one selected from ethylenediamine, triethylamine, diisopropylethylamine, pyridine, and piperidine; preferably pyridine. The addition time of the organic base reagent a should be controlled within 2 hours to avoid more impurities during the reaction and bring difficulties to subsequent purification.

[0021] According to an embodiment of the present invention, the molar amount of the organic base reagent a is equivalent to 0.6 to 1.5 times the molar amount of the long-chain fatty diacid; preferably 1.0 to 1.2 times; more preferably 1.04 to 1.05 times.

[0022] According to an embodiment of the present invention, the temperature at which the long-chain fatty diacyl chloride contacts with tert-butanol and the organic base reagent a in step S2 is 20 °C to 30 °C; preferably 20 °C to 25 °C.

[0023] According to an embodiment of the present invention, the contact time of the long-chain fatty diacyl chloride with tert-butanol and the organic base reagent a in step S2 is 2 to 16 hours; preferably 2 to 4 hours; more preferably 3 hours.

[0024] According to an embodiment of the present invention, step S2 further includes contacting the long-chain fatty diacyl chloride with tert-butanol and the organic base reagent a to obtain a mixed solution, dissolving the mixed solution in a first solvent, and collecting the precipitate;

[0025] The first solvent includes at least one selected from N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran; preferably N,N-dimethylformamide.

[0026] According to an embodiment of the present invention, the temperature of the first solvent is 0 to 4 °C. This is conducive to controlling the impurities generated during the operation.

[0027] According to an embodiment of the present invention, the volume fraction of N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran in the first solvent is 16.7% to 50%; preferably 25% to 33.3%; more preferably 33.3%.

[0028] According to an embodiment of the present invention, the volume-to-mass ratio of the first solvent to the long-chain aliphatic diacid is (10 - 50 mL) : 1 g; preferably (20 - 30 mL) : 1 g; more preferably 30 mL : 1 g.

[0029] According to an embodiment of the present invention, step S2 further includes washing and recrystallizing the precipitate.

[0030] According to an embodiment of the present invention, the washing treatment is carried out in at least one second solvent selected from dichloromethane, ethyl acetate, petroleum ether, and n-heptane; preferably petroleum ether. This increases the purity of the product, and the corresponding number of washing times can be multiple.

[0031] According to an embodiment of the present invention, the volume-to-mass ratio of the second solvent to the long-chain aliphatic diacid is (10 - 50 mL) : 1 g; preferably (10 - 20 mL) : 1 g.

[0032] According to an embodiment of the present invention, the recrystallization treatment is carried out in a third solvent; the third solvent is petroleum ether or n-heptane; preferably n-heptane.

[0033] According to an embodiment of the present invention, the volume-to-mass ratio of the third solvent to the long-chain aliphatic diacid is 4 mL : 1 g.

[0034] The second aspect of the present invention provides a method for preparing a long-chain aliphatic diacid derivative, comprising the following steps:

[0035] (1) Prepare the long-chain aliphatic diacid mono-tert-butyl ester by the method according to any one of claims 1 to 3 for the long-chain aliphatic diacid, and the long-chain aliphatic diacid has the structure shown in formula (A);

[0036] (2) Carry out an esterification reaction between the long-chain aliphatic diacid mono-tert-butyl ester and N-hydroxysuccinimide to obtain the long-chain aliphatic diacid succinimide tert-butyl ester;

[0037] (3) The long-chain fatty acid succinimidyl tert-butyl ester is subjected to a nucleophilic addition amidation reaction with the compound shown by formula (B) to obtain the compound shown by formula (C);

[0038] (4) The compound shown by formula (C) is subjected to a hydrogenation reaction to obtain the compound shown by formula (D);

[0039] (5) The compound shown by formula (D) is subjected to a nucleophilic addition amidation reaction with the compound shown by formula (E) to obtain the compound shown by formula (F);

[0040] (6) The compound shown by formula (F) is subjected to an esterification reaction with N-hydroxysuccinimide to obtain the compound shown by formula (G);

[0041] (7) The compound shown by formula (G) is subjected to a nucleophilic addition amidation reaction with the compound shown by formula (H) to obtain the compound shown by formula (I);

[0042] (8) The compound shown by formula (I) is subjected to an esterification reaction with N-hydroxysuccinimide to obtain the compound shown by formula (J);

[0043] (9) The compound shown by formula (J) is subjected to a deprotection reaction to obtain the compound shown by formula (K);

[0044]

[0045]

[0046] Wherein, X is an integer from 6 to 32; Y is an integer from 2 to 8; Z is an integer from 1 to 12.

[0047] The present invention provides a method for preparing a fatty side chain, which has the advantages of simple operation, low content of product-related impurities, easy purification of intermediates, high yield, simple product purification, and commercialization, and can be used for preparing insulin derivatives with high purity and high yield.

[0048] According to an embodiment of the present invention, the esterification reaction in step (2) is carried out in at least one fourth solvent of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, acetonitrile, and water at -10°C to 20°C for 2 hours and then continued to react at 15°C to 40°C for 3 to 24 hours.

[0049] According to an embodiment of the present invention, the volume-mass ratio of the fourth solvent to the long-chain fatty acid monoter-butyl ester is (5 - 20 mL):1 g.

[0050] According to an embodiment of the present invention, step (2) further includes contacting the long-chain fatty acid monoter-butyl ester with dicyclohexylcarbodiimide.

[0051] According to an embodiment of the present invention, the molar amount of the N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the mono-tert-butyl long-chain aliphatic diacid ester.

[0052] According to an embodiment of the present invention, the molar amount of the dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the mono-tert-butyl long-chain aliphatic diacid ester.

[0053] According to an embodiment of the present invention, step (2) further includes filtering, crystallization, and washing the system after the esterification reaction.

[0054] According to an embodiment of the present invention, the washing treatment is carried out in at least one fifth solvent selected from methanol, ethanol, isopropanol, tert-butanol, n-hexane, n-octane, and cyclohexane.

[0055] According to an embodiment of the present invention, the volume-mass ratio of the fifth solvent to the mono-tert-butyl long-chain aliphatic diacid ester is (1 to 3 mL): 1 g.

[0056] According to an embodiment of the present invention, the nucleophilic addition amidation reaction in step (3) is carried out in at least one sixth solvent selected from chloroform, N,N-dimethylformamide, dimethylacetamide, dimethyl sulfoxide, dichloromethane, and water at 20°C to 40°C.

[0057] According to an embodiment of the present invention, the volume-mass ratio of the sixth solvent to the tert-butyl succinimide long-chain aliphatic diacid ester is (5 to 20 mL): 1 g.

[0058] According to an embodiment of the present invention, the molar amount of the compound represented by formula (B) is equivalent to 1.0 to 1.8 times the molar amount of the tert-butyl succinimide long-chain aliphatic diacid ester.

[0059] According to an embodiment of the present invention, step (3) further includes contacting the tert-butyl succinimide long-chain aliphatic diacid ester with at least one organic base reagent b selected from 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, piperidine, pyridine, ethanolamine, ethylenediamine, and triethylamine.

[0060] According to an embodiment of the present invention, the molar amount of the organic base reagent b is equivalent to 1.5 to 3.0 times the molar amount of the tert-butyl succinimide long-chain aliphatic diacid ester.

[0061] According to an embodiment of the present invention, the hydrogenation reaction in step (4) is carried out in at least one seventh solvent selected from acetonitrile, methanol, acetone, tetrahydrofuran, ethanol, dichloromethane, and chloroform, and reacts with hydrogen at 20°C to 35°C for 1 to 3 hours.

[0062] According to an embodiment of the present invention, the volume-to-mass ratio of the seventh solvent to the compound represented by the formula (C) is (5 - 20 mL) : 1 g.

[0063] According to an embodiment of the present invention, the hydrogenation reaction is carried out in the presence of a catalyst.

[0064] According to an embodiment of the present invention, the catalyst is Pd / C.

[0065] According to an embodiment of the present invention, the mass ratio of the catalyst to the compound represented by the formula (C) is (0.05 - 0.15) : 100.

[0066] According to an embodiment of the present invention, in step (5), the nucleophilic addition amidation reaction is carried out in at least one eighth solvent selected from dichloromethane, ethyl acetate, acetone, methanol, and acetonitrile at a temperature of 25°C to 40°C.

[0067] According to an embodiment of the present invention, the volume-to-mass ratio of the eighth solvent to the compound represented by the formula (D) is (5 - 20 mL) : 1 g.

[0068] According to an embodiment of the present invention, the molar amount of the compound represented by the formula (E) is equivalent to 1.0 - 1.8 times the molar amount of the compound represented by the formula (D).

[0069] According to an embodiment of the present invention, step (5) further includes contacting the compound represented by the formula (D) with at least one organic base reagent c selected from sodium hydroxide, potassium tert-butoxide, triethylamine, 4-dimethylaminopyridine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0070] According to an embodiment of the present invention, the molar amount of the organic base reagent c is equivalent to 2.0 - 3.0 times the molar amount of the compound represented by the formula (D).

[0071] According to an embodiment of the present invention, step (5) further includes subjecting the system after the nucleophilic addition amidation reaction to crystallization and washing treatments.

[0072] According to an embodiment of the present invention, the washing treatment is carried out in at least one ninth solvent selected from isopropanol, n-heptane, ethanol, methanol, cyclohexane, ethyl acetate, acetonitrile, n-hexane, petroleum ether, and diethyl ether.

[0073] According to an embodiment of the present invention, the volume-to-mass ratio of the ninth solvent to the compound represented by the formula (D) is (3 - 20 mL) : 1 g.

[0074] According to an embodiment of the present invention, the esterification reaction in step (6) is carried out in at least one of the tenth solvents including dichloromethane, methyl tert-butyl ether, 1,4-dioxane, tetrahydrofuran, water, toluene, and chlorobenzene, at a temperature of -10°C to 10°C for 2 hours, and then continued to react at 15 - 40°C for 3 - 24 hours.

[0075] According to an embodiment of the present invention, the volume-to-mass ratio of the tenth solvent to the compound represented by formula (F) is (5 - 20 mL) : 1 g.

[0076] According to an embodiment of the present invention, the molar amount of N-hydroxysuccinimide is equivalent to 1.0 - 1.8 times the molar amount of the compound represented by formula (F).

[0077] According to an embodiment of the present invention, step (6) further includes contacting the compound represented by formula (F) with dicyclohexylcarbodiimide.

[0078] According to an embodiment of the present invention, the molar amount of dicyclohexylcarbodiimide is equivalent to 1.0 - 1.8 times the molar amount of the compound represented by formula (F).

[0079] According to an embodiment of the present invention, step (6) further includes filtering, crystallization, and washing the system after the esterification reaction.

[0080] According to an embodiment of the present invention, the washing treatment is carried out in at least one of the eleventh solvents including ether, ethanol, ethyl acetate, isopropyl acetate, petroleum ether, isopropanol, n-heptane, and n-octane.

[0081] According to an embodiment of the present invention, the volume-to-mass ratio of the eleventh solvent to the compound represented by formula (F) is (3 - 20 mL) : 1 g.

[0082] According to an embodiment of the present invention, the nucleophilic addition amidation reaction in step (7) is carried out in at least one of the twelfth solvents including dichloromethane, ethanol, acetonitrile, N,N-dimethylformamide, and acetone, at a temperature of 25°C to 40°C.

[0083] According to an embodiment of the present invention, the volume-to-mass ratio of the twelfth solvent to the compound represented by formula (G) is (5 - 20 mL) : 1 g.

[0084] According to an embodiment of the present invention, step (7) further includes contacting the compound represented by formula (G) with at least one of the organic base reagents d including triethylamine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, tetramethylhexamethylenediamine, and piperidine.

[0085] According to an embodiment of the present invention, the molar amount of the organic base reagent d is equivalent to 2.0 to 3.0 times the molar amount of the compound represented by the formula (G).

[0086] According to an embodiment of the present invention, step (7) further includes subjecting the system after the nucleophilic addition amidation reaction to crystallization and washing treatments.

[0087] According to an embodiment of the present invention, the washing treatment is carried out in at least one thirteenth solvent selected from petroleum ether, n-hexane, cyclohexane, n-heptane, ethyl acetate, isopropanol, and ethanol.

[0088] According to an embodiment of the present invention, the volume-mass ratio of the thirteenth solvent to the compound represented by the formula (G) is (3 - 20 mL): 1 g.

[0089] According to an embodiment of the present invention, the molar amount of the compound represented by the formula (H) is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by the formula (G).

[0090] According to an embodiment of the present invention, the esterification reaction in step (8) is carried out in at least one fourteenth solvent selected from dichloromethane, tetrahydrofuran, acetonitrile, p-xylene, o-xylene, and diethyl ether, at -10°C to 10°C for 2 hours, and then continued to react at 15 - 40°C for 3 - 24 hours.

[0091] According to an embodiment of the present invention, the volume-mass ratio of the fourteenth solvent to the compound represented by the formula (I) is (5 - 20 mL): 1 g.

[0092] According to an embodiment of the present invention, the molar amount of N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by the formula (I).

[0093] According to an embodiment of the present invention, step (8) further includes contacting the compound represented by the formula (I) with dicyclohexylcarbodiimide.

[0094] According to an embodiment of the present invention, the molar amount of dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by the formula (I).

[0095] According to an embodiment of the present invention, step (8) further includes subjecting the system after the esterification reaction to filtration, crystallization, and washing treatments.

[0096] According to an embodiment of the present invention, the washing treatment is carried out in at least one fifteenth solvent selected from isopropanol, ethanol, tert-butanol, n-heptane, n-hexane, and n-octane.

[0097] According to an embodiment of the present invention, the volume-to-mass ratio of the fifteenth solvent to the compound represented by the formula (I) is (3 - 20 mL): 1 g.

[0098] According to an embodiment of the present invention, in step (9), the deprotection reaction is carried out by contacting with trifluoroacetic acid in at least one sixteenth solvent selected from ethyl acetate, isopropanol, ethanol, chloroform, dichloromethane, and N,N-dimethylformamide under the condition of 0 - 20 °C.

[0099] According to an embodiment of the present invention, the volume-to-mass ratio of the sixteenth solvent to the compound represented by the formula (J) is (5 - 20 mL): 1 g.

[0100] According to an embodiment of the present invention, the volume-to-mass ratio of the trifluoroacetic acid to the compound represented by the formula (J) is (5 - 10 mL): 1 g.

[0101] According to an embodiment of the present invention, step (9) further includes dissolving the system after the deprotection reaction in at least one seventeenth solvent selected from p-xylene, chlorobenzene, toluene, tetrahydrofuran, and ether and performing a reduced-pressure distillation operation.

[0102] According to an embodiment of the present invention, the volume-to-mass ratio of the seventeenth solvent to the compound represented by the formula (J) is (5 - 20 mL): 1 g.

[0103] According to an embodiment of the present invention, Y is 2, 4, 6, or 8; preferably 4.

[0104] According to an embodiment of the present invention, the structure of the compound represented by the formula (B) is

[0105] According to an embodiment of the present invention, the structure of the compound represented by the formula (C) is

[0106] According to an embodiment of the present invention, the structure of the compound represented by the formula (D) is

[0107] According to an embodiment of the present invention, Z is an integer from 2 to 6; preferably 2.

[0108] According to an embodiment of the present invention, the structure of the compound represented by the formula (F) is

[0109] According to an embodiment of the present invention, the structure of the compound represented by the formula (G) is

[0110] According to an embodiment of the present invention, the structure of the compound represented by formula (I) is

[0111] According to an embodiment of the present invention, the structure of the compound represented by formula (J) is

[0112] The third aspect of the present invention provides the use of the method described in the second aspect in the preparation of insulin analogs, GLP-1 analogs, GLP-1 / GIP analogs or GLP-1 / GIP / GCG analogs.

[0113] The method provided by the second aspect of the present invention has the following advantages: (1) The raw materials are cheap and easily available, and the preparation cost is low; (2) The reaction conditions are simple, the process flow is stable, the reaction time is short, the product purification is simple, and the operation is convenient; (3) Good chemical selectivity, high product yield, etc., so it can be used to prepare insulin derivatives with high purity and high yield.

[0114] The fourth aspect of the present invention provides a method for preparing a hypoglycemic protein drug, the method comprising: modifying a protein with a long fatty acid chain diacid derivative to obtain the hypoglycemic protein drug; wherein the long fatty acid chain diacid derivative is obtained according to the method described in the second aspect.

[0115] According to an embodiment of the present invention, the hypoglycemic protein drug comprises at least one selected from insulin analogs, GLP-1 analogs, GLP-1 / GIP analogs, GLP-1 / GIP / GCG analogs.

[0116] According to an embodiment of the present invention, the protein is Icodec insulin, liraglutide, semaglutide or tirzepatide.

[0117] According to an embodiment of the present invention, the modification is a fatty side chain modification.

[0118] The additional aspects and advantages of the present invention will be given in part in the following description, will become apparent in part from the following description, or will be understood through the practice of the present invention. Detailed Description of the Invention

[0119] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0120] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more.

[0121] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0122] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0123] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0124] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may but do not necessarily occur, and this description includes the cases where such events or conditions occur and the cases where such events or conditions do not occur.

[0125] According to an embodiment of the present invention, the preparation method of the long-chain fatty diacid derivative provided by the present invention can be described as follows:

[0126] (1) React a long-chain fatty diacid shown in Formula A with thionyl chloride to obtain an acyl chloride with two chlorine groups shown in Formula A(1); then react the acyl chloride with tert-butanol to obtain an ester with one chlorine group shown in Formula A(2); then replace the chlorine group with a hydroxyl group to obtain a long-chain fatty diacid mono-tert-butyl ester shown in Formula A(3); wherein, X in the following reaction equation is an integer from 6 to 32;

[0127] The specific steps may be as follows: reacting a long-chain aliphatic diacid with thionyl chloride to prepare an acyl chloride, with the reaction conditions being heating under reflux at 80 °C for 3 hours or reacting overnight at 15 - 30 °C under the catalysis of DMF. When choosing the reaction conditions of reacting overnight at 15 - 30 °C under the catalysis of DMF, it is preferable to resuspend the long-chain aliphatic diacid in dichloromethane, then add DMF and thionyl chloride, and react overnight at 15 - 30 °C. In addition, the amount of thionyl chloride used is slightly in excess, and the unreacted thionyl chloride is removed by vacuum distillation, and dichloromethane is added to dissolve the acyl chloride. The organic base reagent a is dropwise added to the mixture of the acyl chloride and tert-butanol, and a chloro-group-containing ester is formed by reaction. After removing the solvent by vacuum distillation, a mixed solution of a first solvent pre-cooled to 0 - 4 °C and water is added, and the precipitate is collected by filtration; the solvent in the precipitate is removed, and then a second solvent is added to the precipitate for extraction. After evaporating the solvent, recrystallization is carried out to obtain a long-chain aliphatic diacid mono-tert-butyl ester.

[0128] Among them, the organic base reagent a includes at least one selected from ethylenediamine, triethylamine, diisopropylethylamine, pyridine, and piperidine; preferably pyridine;

[0129] The molar amount of the organic base reagent a is equivalent to 0.6 - 1.5 times the molar amount of the long-chain aliphatic diacid; preferably 1.0 - 1.2 times; more preferably 1.04 - 1.05 times;

[0130] The first solvent includes at least one selected from N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran; preferably N,N-dimethylformamide;

[0131] The volume fraction of N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran in the first solvent is 16.7% - 50%; preferably 25% - 33.3%; more preferably 33.3%; the volume-mass ratio of the first solvent to the long-chain aliphatic diacid is (10 - 50 mL):1 g; preferably (20 - 30 mL):1 g; more preferably 30 mL:1 g;

[0132] The second solvent is at least one of dichloromethane, ethyl acetate, petroleum ether, and n-heptane;

[0133] The volume-mass ratio of the second solvent to the long-chain aliphatic diacid is (10 - 50 mL):1 g; preferably (10 - 20 mL):1 g;

[0134] The recrystallization operation is carried out in a third solvent, and the third solvent is one or two of petroleum ether or n-heptane, preferably n-heptane;

[0135] The volume-mass ratio of the third solvent to the long-chain aliphatic diacid is 4 mL:1 g.

[0136]

[0137] (2) Esterify the long-chain fatty diacid monoter-butyl ester with N-hydroxysuccinimide to obtain the succinimidyl ter-butyl ester of the long-chain fatty diacid;

[0138] The specific steps may be as follows: Dissolve the long-chain fatty diacid monoter-butyl ester in a fourth solvent, cool the temperature to -10°C to 20°C, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, continue the reaction at -10°C to 20°C for 2 hours, restore the temperature to 15 - 40°C and continue the reaction for 3 - 24 hours, filter to remove the precipitate, concentrate by reduced pressure distillation to dryness to obtain a solid, perform recrystallization, and wash the filter cake with a fifth solvent to obtain the succinimidyl ter-butyl ester of the long-chain fatty diacid.

[0139]

[0140] Among them, the fourth solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, acetonitrile, and water;

[0141] The volume-mass ratio of the fourth solvent to the long-chain fatty diacid monoter-butyl ester is (5 - 20 mL):1 g;

[0142] The molar amount of the N-hydroxysuccinimide is equivalent to 1.0 - 1.8 times the molar amount of the long-chain fatty diacid monoter-butyl ester;

[0143] The molar amount of the N-hydroxysuccinimide is equivalent to 1.0 - 1.8 times the molar amount of the long-chain fatty diacid monoter-butyl ester;

[0144] The fifth solvent is at least one of methanol, ethanol, isopropanol, tert-butanol, n-hexane, n-octane, and cyclohexane;

[0145] The volume-mass ratio of the fifth solvent to the long-chain fatty diacid monoter-butyl ester is (1 - 3 mL):1 g;

[0146] (3) Perform a nucleophilic addition amidation reaction between the succinimidyl ter-butyl ester of the long-chain fatty diacid and the compound shown in formula (B) to obtain the compound shown in formula (C);

[0147] The specific steps may be as follows: Dissolve the succinimidyl ter-butyl ester of the long-chain fatty diacid and N6-((benzyloxy)carbonyl)-L-lysine ter-butyl ester in a sixth solvent, place it under stirring overnight at 25 - 40°C in the presence of an organic base reagent b. After the reaction is completed, quench the reaction successively with a 0.5 - 2.0 mol / L hydrochloric acid solution and water, the volume ratio of the hydrochloric acid solution to water is 1:5 - 1:10, cool the temperature to -10°C to 10°C and stir for crystallization, filter and wash the filter cake with an appropriate amount of water until neutral to obtain the ter-butyl ester of long-chain fatty diacyl-N6-((benzyloxy)carbonyl)-L-lysine ter-butyl ester;

[0148]

[0149] Among them, the sixth solvent is chloroform, N,N-dimethylformamide, dimethylacetamide, dimethyl sulfoxide, dichloromethane, water;

[0150] The volume-mass ratio of the sixth solvent to the long fatty chain diacid succinimide tert-butyl ester is (5 - 20 mL):1 g;

[0151] The molar amount of N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester is equivalent to 1.0 - 1.8 times the molar amount of the long fatty chain diacid succinimide tert-butyl ester;

[0152] The organic base reagent b is at least one of 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, piperidine, pyridine, ethanolamine, ethylenediamine, triethylamine;

[0153] The molar amount of the organic base reagent b is equivalent to 1.5 - 3.0 times the molar amount of the long fatty chain diacid succinimide tert-butyl ester;

[0154] (4) Subject the compound shown by the formula (C) to a hydrogenation reaction to obtain the compound shown by the formula (D);

[0155] The specific steps can be: Dissolve tert-butyl ester long fatty chain diacyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester in the seventh solvent, add Pd / C, then dropwise add 0.1 mL of trifluoroacetic acid. After replacing the atmosphere in the system with hydrogen three times, put on a hydrogen balloon and stir at 20°C - 35°C for 1 - 3 hours. After the reaction is completed, filter to remove Pd / C, and rotary evaporate the filtrate to remove the solvent.

[0156] After drying, tert-butyl ester long fatty chain diacyl L-lysine tert-butyl ester is obtained.

[0157]

[0158] Among them, the seventh solvent is acetonitrile, methanol, acetone, tetrahydrofuran, ethanol, dichloromethane, chloroform;

[0159] The volume-mass ratio of the seventh solvent to the tert-butyl ester long fatty chain diacyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester is (5 - 20 mL):1 g;

[0160] The mass ratio of the catalyst Pd / C to the tert-butyl ester long fatty chain diacyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester is (0.05 - 0.15):100;

[0161] (5) Carry out a nucleophilic addition amidation reaction between the compound represented by formula (D) and the compound represented by formula (E) to obtain the compound represented by formula (F);

[0162] Specific steps may be as follows: Add tert-butyl ester long fatty chain-diacyl L-lysine tert-butyl ester and succinic anhydride into the eighth solvent, and stir overnight at 25 °C to 40 °C in the presence of organic base reagent c. After the reaction is completed, quench the reaction successively with 0.5 - 2.0 mol / L hydrochloric acid solution and water, where the volume ratio of the hydrochloric acid solution to water is 1:5 - 1:10. Extract, collect the organic phase, concentrate it to dryness under reduced pressure distillation to obtain a solid, recrystallize, filter, and wash with the ninth solvent. Obtain tert-butyl ester long fatty chain diacyl L-lysine-diacyl-tert-butyl ester.

[0163]

[0164] Among them, the eighth solvent is dichloromethane, ethyl acetate, acetone, methanol, acetonitrile;

[0165] The volume-mass ratio of the eighth solvent to the tert-butyl ester long fatty chain-diacyl L-lysine tert-butyl ester is (5 - 20 mL):1 g;

[0166] The molar amount of the succinic anhydride is equivalent to 1.0 - 1.8 times the molar amount of the tert-butyl ester long fatty chain-diacyl L-lysine tert-butyl ester;

[0167] The organic base reagent c is at least one of sodium hydroxide, potassium tert-butoxide, triethylamine, 4-dimethylaminopyridine, pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene;

[0168] The molar amount of the organic base reagent c is equivalent to 2.0 - 3.0 times the molar amount of the tert-butyl ester long fatty chain-diacyl L-lysine tert-butyl ester;

[0169] The ninth solvent is isopropanol, n-heptane, ethanol, methanol, cyclohexane, ethyl acetate, acetonitrile, n-hexane, petroleum ether, diethyl ether;

[0170] The volume-mass ratio of the ninth solvent to the tert-butyl ester long fatty chain-diacyl L-lysine tert-butyl ester is (3 - 20 mL):1 g;

[0171] (6) Carry out an esterification reaction between the compound represented by formula (F) and N-hydroxysuccinimide to obtain the compound represented by formula (G);

[0172] The specific steps may be as follows: Dissolve the long fatty chain-diacyl L-lysine-diacyl-tert-butyl ester obtained in step (5) in the tenth solvent, cool down to -10°C to 10°C, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, and continue the reaction at -10°C to 10°C for 2 hours. Then, warm it up to 15°C to 40°C and continue the reaction for 3 to 24 hours. Filter to remove the precipitate, concentrate it to dryness by vacuum distillation to obtain a solid, perform recrystallization, filter, and wash it with the eleventh solvent to obtain long fatty chain-diacyl L-lysine-diacyl-OSu-tert-butyl ester.

[0173]

[0174] Among them, the tenth solvent is at least one of dichloromethane, methyl tert-butyl ether, 1,4-dioxane, tetrahydrofuran, water, toluene, and chlorobenzene;

[0175] The volume-mass ratio of the tenth solvent to the long fatty chain-diacyl L-lysine-diacyl-tert-butyl ester is (5 - 20 mL) : 1 g;

[0176] The molar amount of N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain-diacyl L-lysine-diacyl-tert-butyl ester;

[0177] The molar amount of dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain-diacyl L-lysine-diacyl-tert-butyl ester;

[0178] The eleventh solvent is at least one of ether, ethanol, ethyl acetate, isopropyl acetate, petroleum ether, isopropanol, n-heptane, and n-octane;

[0179] The volume-mass ratio of the eleventh solvent to the long fatty chain-diacyl L-lysine-diacyl-tert-butyl ester is (3 - 20 mL) : 1 g;

[0180] (7) Perform a nucleophilic addition amidation reaction on the compound shown in formula (G) and the compound shown in formula (H) to obtain the compound shown in formula (I);

[0181] The specific steps may be as follows: Dissolve the long fatty chain-diacyl-L-lysine-diacyl-OSu-tert-butyl ester in the twelfth solvent, add 17-amino-10-oxo-3,6,12,15-tetraoxo-9-azanonanoic acid and the organic base reagent d, and place it for overnight stirring at 25°C to 40°C. After the reaction is completed, quench the reaction successively with 0.5 to 2.0 mol / L hydrochloric acid solution and water, where the volume ratio of the hydrochloric acid solution to water is 1:5 to 1:10. After liquid separation, collect the organic phase, concentrate it by reduced pressure distillation to dryness to obtain a solid, perform recrystallization, filtration, and wash it with the thirteenth solvent. Obtain long fatty chain-diacyl-L-lysine-diacyl-tert-butyl ester-2xOEG-OH.

[0182]

[0183] Among them, the twelfth solvent is at least one of dichloromethane, ethanol, acetonitrile, N,N-dimethylformamide, and acetone;

[0184] The volume-mass ratio of the twelfth solvent to the long fatty chain-diacyl-L-lysine-diacyl-OSu-tert-butyl ester is (5 to 20 mL):1 g;

[0185] The organic base reagent d is at least one of triethylamine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, tetramethylhexanediamine, and piperidine;

[0186] The molar amount of the organic base reagent d is equivalent to 2.0 to 3.0 times the molar amount of the long fatty chain-diacyl-L-lysine-diacyl-OSu-tert-butyl ester;

[0187] The thirteenth solvent is at least one of petroleum ether, n-hexane, cyclohexane, n-heptane, ethyl acetate, isopropanol, and ethanol;

[0188] The volume-mass ratio of the thirteenth solvent to the long fatty chain-diacyl-L-lysine-diacyl-OSu-tert-butyl ester is (3 to 20 mL):1 g;

[0189] The molar amount of the 17-amino-10-oxo-3,6,12,15-tetraoxo-9-azanonanoic acid is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain-diacyl-L-lysine-diacyl-OSu-tert-butyl ester;

[0190] (8) Carry out an esterification reaction between the compound shown in the formula (I) and N-hydroxysuccinimide to obtain the compound shown in the formula (J);

[0191] The specific steps may be as follows: Dissolve the long fatty chain-diacyl L-lysine diacyl-tert-butyl ester-2xOEG-OH obtained in step (7) in the fourteenth solvent, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, and continue the reaction at -10°C to 10°C for 2 hours. Then, restore the temperature to 15°C to 40°C and continue the reaction for 3 to 24 hours. Filter to remove the precipitate, concentrate the filtrate by vacuum distillation to dryness to obtain a solid, perform recrystallization, filter, and wash with solvent n to obtain long fatty chain diacyl L-lysine-diacyl-tert-butyl ester-2xOEG-Osu.

[0192]

[0193] Among them, the fourteenth solvent is at least one of dichloromethane, tetrahydrofuran, acetonitrile, p-xylene, o-xylene, and diethyl ether;

[0194] The volume-mass ratio of the fourteenth solvent to the long fatty chain-diacyl L-lysine diacyl-tert-butyl ester-2xOEG-OH is (5 - 20 mL):1 g;

[0195] The molar amount of N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain-diacyl L-lysine diacyl-tert-butyl ester-2xOEG-OH;

[0196] The molar amount of dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain-diacyl L-lysine diacyl-tert-butyl ester-2xOEG-OH;

[0197] The fifteenth solvent is at least one of isopropanol, ethanol, tert-butanol, n-heptane, n-hexane, and n-octane;

[0198] The volume-mass ratio of the fifteenth solvent to the long fatty chain-diacyl L-lysine diacyl-tert-butyl ester-2xOEG-OH is (3 - 20 mL):1 g;

[0199] (9) Perform a deprotection reaction on the compound shown in formula (J) to obtain the compound shown in formula (K);

[0200] The specific steps may be as follows: Dissolve the long fatty chain diacyl-L-lysine-diacyl-tert-butyl ester-2xOEG-Osu obtained in step (8) in the sixteenth solvent, add trifluoroacetic acid, and perform a de-Boc reaction at 0 to 20°C. After the reaction is completed, concentrate the reaction solution by vacuum distillation to dryness to obtain a crude liquid product. Add an appropriate amount of the seventeenth solvent and continue vacuum distillation to remove the excess trifluoroacetic acid. After obtaining the crude liquid product, add the seventeenth solvent again and perform vacuum distillation. Repeat this process three times. Finally, obtain long fatty chain diacyl-L-lysine-diacyl-2xOEG-OSu.

[0201]

[0202] Among them, the sixteenth solvent is at least one of ethyl acetate, isopropanol, ethanol, chloroform, dichloromethane, and N,N-dimethylformamide;

[0203] The volume-mass ratio of the sixteenth solvent to the long fatty acid chain diacyl-L-lysine-diacyl-tert-butyl ester-2xOEG-Osu is (5-20 mL):1 g;

[0204] The volume-mass ratio of the trifluoroacetic acid to the long fatty acid chain diacyl-L-lysine-diacyl-tert-butyl ester-2xOEG-Osu is (5-10 mL):1 g;

[0205] The seventeenth solvent is at least one of p-xylene, chlorobenzene, toluene, tetrahydrofuran, and ether;

[0206] The volume-mass ratio of the seventeenth solvent to the long fatty acid chain diacyl-L-lysine-diacyl-tert-butyl ester-2xOEG-Osu is (5-20 mL):1 g.

[0207] The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0208] Example 1

[0209] Synthesis of monoter-butyl octadecanedioate

[0210]

[0211] Octadecanedioic acid (40.0 g, 127.2 mmol) was suspended in thionyl chloride (175.5 mL, 2065 mmol), and the mixture was heated under reflux at 80 °C for 3 hours. The excess thionyl chloride was removed by distillation under reduced pressure. Dichloromethane (200 mL) was added to dissolve the residue, and then tert-butanol (14.60 mL, 152.64 mmol) and pyridine (10.8 mL, 133.56 mmol) were added. Pyridine was added dropwise within 2 hours, and the reaction was carried out at 25 °C for 3 h. The solvent was evaporated under reduced pressure, and a mixed solvent of DMF / water (1000 mL, DMF and water were mixed at a volume ratio of 1:2) pre-cooled to 0 - 4 °C was added. The precipitate was collected by filtration. After vacuum drying to constant weight, the product was resuspended in dichloromethane (500 mL), and the filtrate was collected and the solvent was evaporated under reduced pressure. Then it was resuspended in petroleum ether (700 mL), the filtrate was collected and the solvent was evaporated under reduced pressure. Recrystallization was carried out with n-heptane (150 mL), the precipitate was collected by filtration, and after drying under reduced pressure to constant weight, tert-butyl octadecanedioate was obtained. The yield was 15.56 g, and the yield rate was 33%. The purity detected by HPLC was 97.12%.

[0212] MS test of tert-butyl octadecanedioate: ESI-MS m / z: 370.84 [M+H] + , which is in line with the theoretical value.

[0213] Example 2

[0214] Synthesis of tert-butyl octadecanedioate-OSu

[0215]

[0216] Tert-butyl octadecanedioate (10 g, 26.98 mmol), N-hydroxysuccinimide (3.7 g, 32.38 mmol) and 100 mL of DCM were added to a 250 mL single-necked flask. The temperature was lowered to -10 °C, and dicyclohexylcarbodiimide (6.7 g, 32.58 mmol) was added and the reaction continued for 2 hours. Then it was restored to room temperature and stirred for another 10 hours. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated by distillation under reduced pressure to dryness to obtain a crude solid product. Recrystallization was carried out with 80 mL of isopropanol and 80 mL of n-hexane, filtered, and the filter cake was washed with 20 mL of n-hexane. After drying under reduced pressure to constant weight, 10.6 g of white solid tert-butyl octadecanedioate-OSu was obtained. The yield rate was 84.0%, and the purity detected by HPLC was 96.4%.

[0217] MS test of tert-butyl octadecanedioate-OSu: ESI-MS m / z: 467.37 [M+H] + , which is in line with the theoretical value.

[0218] Example 3

[0219] Synthesis of tert-Butyl Ester-Octadecanedioyl-N6-((Benzyloxy)Carbonyl)-L-Lysine tert-Butyl Ester

[0220]

[0221] Add tert-butyl octadecanedioate-OSu (10 g, 21.38 mmol), N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester (7.91 g, 23.52 mmol) and 160 mL of acetonitrile into a 250 mL single-necked flask, place it in an oil bath at 30 °C, add triethylamine (4.32 g, 42.76 mmol), and continue the reaction for 12 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 90 mL of isopropanol and 30 mL of n-hexane for recrystallization, filter, and wash the filter cake with 30 mL of n-hexane, and dry under reduced pressure to constant weight. Obtain 12.91 g of white solid of tert-butyl ester-octadecanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester, with a yield of 87.6%, and the HPLC purity for inspection is 95.1%.

[0222] MS test of tert-butyl ester-octadecanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester: ESI-MS m / z: 689.58 [M+H] + , which is in line with the theoretical value.

[0223] Example 4

[0224] Synthesis of tert-Butyl Ester-Octadecanedioyl-L-Lys-tert-Butyl Ester

[0225]

[0226] Add tert-butyl ester-octadecanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester (10 g, 14.51 mmol) and THF (100 mL) into a 250 mL single-necked reaction flask, add 15% Pd / C (1.5 g) and 0.1 mL of trifluoroacetic acid, replace the system with hydrogen three times, then put on a hydrogen balloon, and stir at 30 °C for 4 hours. After the reaction is completed, filter Pd / C, and concentrate the filtrate under reduced pressure to dryness. Obtain 6.9 g of crude product. The yield is 85.7%, and the HPLC purity for inspection is 97.1%.

[0227] MS test of tert-butyl ester-octadecanedioyl-L-Lys-tert-butyl ester: ESI-MS m / z: 554.49 [M+H] + , which is in line with the theoretical value.

[0228] Example 5

[0229] Synthesis of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl

[0230]

[0231] Add tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester (8 g, 14.42 mmol), succinic anhydride (2.16 g, 21.63 mmol) and 80 mL of THF into a 250 mL single-neck flask, place it in an oil bath at 30 °C, add triethylamine (3.6 g, 36.1 mmol) and continue the reaction for 12 hours. After the reaction is completed, add 2 M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a solid crude product. Add 80 mL of isopropanol and 20 mL of n-hexane for recrystallization, filter, and wash the filter cake with 20 mL of n-hexane, then dry under reduced pressure to constant weight. Obtain 7.9 g of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl, with a yield of 83.7%, and the HPLC purity for submission is 93.58%.

[0232] MS test of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl: ESI-MS m / z: 654.57 [M+H] + , which is in line with the theoretical value.

[0233] Example 6

[0234] Synthesis of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-OSu

[0235]

[0236] Add tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl (10 g, 15.27 mmol), N-hydroxysuccinimide (2.1 g, 18.33 mmol) and 100 mL of DCM into a 250 mL single-neck flask, cool down to -10 °C, add dicyclohexylcarbodiimide (3.8 g, 18.33 mmol) and continue the reaction for 2 hours, then restore to room temperature and continue stirring for 12 hours. After the reaction is completed, filter to remove the precipitate, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a solid crude product. Add 120 mL of ethanol for recrystallization, filter, and wash the filter cake with 30 mL of n-hexane, then dry under reduced pressure to constant weight. Obtain 10.3 g of white solid tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-OSu, with a yield of 89.7%, and the HPLC purity for submission is 96.86%.

[0237] MS test of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-OSu: ESI-MS m / z: 752.27 [M+H]+ , which is in line with the theoretical value.

[0238] Example 7

[0239] Synthesis of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG

[0240]

[0241] Add tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-OSu (10 g, 13.3 mmol), 17-amino-10-oxo-3,6,12,15-tetraoxo-9-azanonanoic acid (5.3 g, 17.3 mmol) and 120 mL of DMF into a 250 mL single-necked flask, place it in an oil bath at 30 °C and stir, add triethylamine (3.3 g, 31.9 mmol) and continue the reaction for 8 hours. After the reaction is completed, add 2M HCl (197.3 g of commercially available 37% hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure to dryness to obtain a solid crude product. Add 120 mL of isopropanol and 30 mL of n-heptane for recrystallization, filter, and wash the filter cake with 15 mL of n-heptane, and dry under reduced pressure to constant weight. 11.8 g of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG was obtained, with a yield of 93.9%, and the HPLC purity for inspection was 90.69%.

[0242] MS test of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG: ESI-MS m / z: 945.68 [M+H] + , which is in line with the theoretical value.

[0243] Example 8

[0244] Synthesis of tert-butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu

[0245]

[0246] tert-Butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG (10 g, 10.5 mmol), N-hydroxysuccinimide (1.7 g, 14.8 mmol) and 100 mL of DCM were added to a 250 mL single-necked flask. The temperature was lowered to -10 °C, and cyclohexylcarbodiimide (3.0 g, 14.8 mmol) was added and the reaction continued for 2 hours. The temperature was then restored to room temperature and stirring continued for 10 hours. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to dryness to obtain a crude solid. The crude solid was recrystallized from 120 mL of ethanol, filtered, and the filter cake was rinsed with 20 mL of petroleum ether and dried under reduced pressure to constant weight. 10.3 g of white solid of tert-Butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu was obtained, with a yield of 94.14% and an HPLC purity of 96.19% for submission for inspection.

[0247] MS test of tert-Butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu: ESI-MS m / z: 1042.93 [M+H] + , which is in line with the theoretical value.

[0248] Example 9

[0249] Synthesis of octadecanedioyl-L-lys-diacyl-2xOEG-OSu

[0250]

[0251] tert-Butyl ester-octadecanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu (10 g, 9.6 mmol), tetrahydrofuran (50 mL), and trifluoroacetic acid (50 mL) were added to a 250 mL single-necked reaction flask and stirred. The mixture was stirred at 0 °C for 3 h. After the reaction was completed, the liquid in the system was distilled to dryness under reduced pressure to obtain a crude yellow liquid. Toluene (50 mL) was added to the crude product to dissolve it. It was then placed on a rotary evaporator again and distilled under reduced pressure to obtain a yellow liquid again. Another 50 mL of toluene was added and it was placed on a rotary evaporator and distilled under reduced pressure again. A yellow liquid was obtained. The above operation was repeated again after adding the solvent and distilling under reduced pressure. Finally, 7.2 g of yellow liquid of octadecanedioyl-L-lys-diacyl-2xOEG-OSu was obtained, with a yield of 80.64% and an HPLC purity of 95.78% for submission for inspection.

[0252] MS test of octadecanedioyl-L-lys-diacyl-2xOEG-OSu: ESI-MS m / z: 930.57 [M+H] + , which is in line with the theoretical value.

[0253] Example 10

[0254] Synthesis of monoter-butyl ester of eicosanedioic acid

[0255]

[0256] Suspend eicosanedioic acid (40.0 g, 116.8 mmol) in thionyl chloride (162 mL, 2219 mmol), heat under reflux at 80 °C for 3 hours, and after the reaction is completed, remove the excess thionyl chloride by distillation under reduced pressure. Add dichloromethane (200 mL) to dissolve, add tert-butanol (14.52 mL, 151.84 mmol) and pyridine (9.9 mL, 122.64 mmol), add pyridine dropwise within 2 hours, and react at 25 °C for 3 h. Evaporate the solvent under reduced pressure, add 900 mL of a pre-cooled DMF / water mixed solvent (DMF and water are mixed in a volume ratio of 1:2), filter to collect the precipitate. Dry in vacuo to constant weight, resuspend in dichloromethane (500 mL), collect the filtrate and evaporate the solvent under reduced pressure, resuspend in petroleum ether (700 mL), collect the filtrate and evaporate under reduced pressure, add n-heptane (150 mL) for recrystallization, filter to collect the precipitate, and dry under reduced pressure to constant weight to obtain tert-butyl eicosanedioate, with a yield of 16.94 g and a yield of 36.3%, and the purity detected by HPLC is 97.8%.

[0257] MS test of tert-butyl eicosanedioate: ESI-MS m / z: 398.68 [M+H] + , which is in line with the theoretical value.

[0258] Example 11

[0259] Synthesis of tert-butyl eicosanedioate-OSu

[0260]

[0261] Add tert-butyl eicosanedioate (10 g, 25.08 mmol), N-hydroxysuccinimide (3.5 g, 30.01 mmol) and 100 mL of DCM to a 250 mL single-necked flask, cool to -10 °C, add dicyclohexylcarbodiimide (6.2 g, 30.01 mmol) and continue to react for 2 hours, then return to room temperature and continue stirring for 6 hours. After the reaction is completed, filter to remove the precipitate, concentrate the filtrate by distillation under reduced pressure to dryness to obtain a solid crude product, add 80 mL of isopropanol and 80 mL of n-hexane for recrystallization, filter, and wash the filter cake with 20 mL of n-hexane, and dry under reduced pressure to constant weight. Obtain 11.6 g of white solid tert-butyl eicosanedioate-OSu, with a yield of 93.5%, and the purity detected by HPLC is 93.96%.

[0262] MS test of tert-butyl eicosanedioate-OSu: ESI-MS m / z: 495.93 [M+H] + , which is in line with the theoretical value.

[0263] Example 12

[0264] Synthesis of tert-Butyl Ester-Docosanedioyl-N6-((Benzyloxy)Carbonyl)-L-Lysine tert-Butyl Ester

[0265]

[0266] Add tert-butyl docosanedioate-OSu (10.0 g, 20.17 mmol), N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester (8.8 g, 26.22 mmol) and 120 mL of THF into a 250 mL single-necked flask, place it in an oil bath at 30 °C and stir. Add triethylamine (4.32 g, 44.37 mmol) and continue the reaction for 12 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, and concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 90 mL of isopropanol and 30 mL of n-hexane for recrystallization, filter, and wash the filter cake with 30 mL of n-hexane, and dry under reduced pressure to constant weight. Obtain 12.91 g of white solid of tert-butyl ester-docosanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester, with a yield of 87.6% and a tested HPLC purity of 95.1%.

[0267] MS test of tert-butyl ester-docosanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester: ESI-MS m / z: 717.34 [M+H] + , consistent with the theoretical value.

[0268] Example 13

[0269] Synthesis of tert-Butyl Ester-Docosanedioyl-L-Lys-tert-Butyl Ester

[0270]

[0271] Add tert-butyl ester-docosanedioyl-N6-((benzyloxy)carbonyl)-L-lysine tert-butyl ester (10 g, 13.95 mmol) and THF (100 mL) into a 250 mL single-necked reaction flask, add 10% Pd / C (1.0 g) and 0.1 mL of trifluoroacetic acid. After replacing the system with hydrogen three times, put on a hydrogen balloon and stir at 30 °C for 4 hours. After the reaction is completed, filter Pd / C and distill the filtrate under reduced pressure to dryness. Obtain 7.1 g of crude product. The yield is 87.3% and the tested HPLC purity is 98.7%.

[0272] MS test of tert-butyl ester-docosanedioyl-L-Lys-tert-butyl ester: ESI-MS m / z: 583.16 [M+H] + , consistent with the theoretical value.

[0273] Example 14

[0274] Synthesis of tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl

[0275]

[0276] Add tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester (15 g, 25.73 mmol), succinic anhydride (3.35 g, 33.45 mmol) and 160 mL of THF into a 250 mL single-neck flask, place it in an oil bath at 30 °C, add triethylamine (3.9 g, 38.6 mmol), and continue the reaction for 12 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 120 mL of isopropanol and 20 mL of n-hexane for recrystallization, filter, and wash the filter cake with 20 mL of n-hexane, then dry under reduced pressure to constant weight. 15.7 g of tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl is obtained, with a yield of 89.4% and the HPLC purity for submission is 91.49%.

[0277] MS test of tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl: ESI-MS m / z: 683.47 [M+H] + , which is in line with the theoretical value.

[0278] Example 15

[0279] Synthesis of tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-OSu

[0280]

[0281] Add tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl (10 g, 14.64 mmol), N-hydroxysuccinimide (1.85 g, 16.1 mmol) and 100 mL of DCM into a 250 mL single-neck flask, cool down to -10 °C, add dicyclohexylcarbodiimide (3.32 g, 16.1 mmol), and continue the reaction for 2 hours. Then restore to room temperature and continue stirring for 12 hours. After the reaction is completed, filter to remove the precipitate, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 120 mL of ethanol for recrystallization, filter, and wash the filter cake with 30 mL of n-hexane, then dry under reduced pressure to constant weight. 10.8 g of white solid of tert-butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-OSu is obtained, with a yield of 94.6% and the HPLC purity for submission is 97.19%.

[0282] tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-OSu MS test: ESI-MS m / z: 780.76 [M+H] + , which is consistent with the theoretical value.

[0283] Example 16

[0284] Synthesis of tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG

[0285]

[0286] Add tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-OSu (10 g, 12.8 mmol), 17-amino-10-oxo-3,6,12,15-tetraoxo-9-azanonanoic acid (4.7 g, 15.4 mmol) and 120 mL of DMF into a 250 mL single-necked flask, place it in an oil bath at 30 °C, add triethylamine (2.8 g, 28.2 mmol) and continue the reaction for 8 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L with water) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 120 mL of isopropanol and 30 mL of n-heptane for recrystallization, filter, and wash the filter cake with 15 mL of n-heptane, and dry under reduced pressure to constant weight. 8.9 g of tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG was obtained, with a yield of 71.2% and the HPLC purity for submission was 95.7%.

[0287] tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG MS test: ESI-MS m / z: 973.61 [M+H] + , which is consistent with the theoretical value.

[0288] Example 17

[0289] Synthesis of tert-Butyl ester-eicosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu

[0290]

[0291] tert-Butyl ester-eicosanedicarbonyl-L-lys-tert-butyl ester-dicarbonyl-2xOEG (8 g, 8.22 mmol), N-hydroxysuccinimide (1.1 g, 9.9 mmol) and 80 mL of DCM were added to a 250 mL single-necked flask and stirred. The temperature was lowered to -10 °C, and dicyclohexylcarbodiimide (2.0 g, 9.9 mmol) was added and the reaction continued for 2 hours. The temperature was then restored to room temperature and stirring continued for 5 hours. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure by distillation to dryness to obtain a crude solid. 80 mL of ethanol was added for recrystallization, and the mixture was filtered. The filter cake was rinsed with 10 mL of petroleum ether and dried under reduced pressure to constant weight. 7.3 g of white solid of tert-Butyl ester-eicosanedicarbonyl-L-lys-tert-butyl ester-dicarbonyl-2xOEG-OSu was obtained, with a yield of 82.95% and the HPLC purity for inspection was 96.69%.

[0292] MS test of tert-Butyl ester-eicosanedicarbonyl-L-lys-tert-butyl ester-dicarbonyl-2xOEG-OSu: ESI-MS m / z: 1070.58 [M+H] + , which is in line with the theoretical value.

[0293] Example 18

[0294] Synthesis of eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG-OSu

[0295]

[0296] tert-Butyl ester-eicosanedicarbonyl-L-lys-tert-butyl ester-dicarbonyl-2xOEG-OSu (8 g, 7.5 mmol), dichloromethane (40 mL), and trifluoroacetic acid (40 mL) were added to a 250 mL single-necked reaction flask, and the mixture was stirred at 0 °C for 3 h. After the reaction was completed, the liquid in the system was concentrated under reduced pressure by distillation to dryness to obtain a crude yellow liquid. Chlorobenzene (40 mL) was added to the crude product to dissolve it. It was then placed on a rotary evaporator again, and after reduced pressure distillation, a yellow liquid was obtained again. 40 mL of chlorobenzene was added again and it was placed on a rotary evaporator for reduced pressure distillation again. A yellow liquid was obtained again. The above operation was repeated once more after adding the solvent and then performing reduced pressure distillation. Finally, 6.3 g of viscous yellow liquid of eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG-OSu was obtained, with a yield of 87.67% and the HPLC purity for inspection was 93.63%.

[0297] MS test of eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG-OSu: ESI-MS m / z: 958.39 [M+H] + , which is in line with the theoretical value.

[0298] Example 19

[0299] Synthesis of Monotert-butyl Docosanedioate

[0300]

[0301] Suspend docosanedioic acid (40.0 g, 107.9 mmol) in thionyl chloride (156 mL, 2158 mmol), heat under reflux at 80 °C for 3 hours, and remove the excess thionyl chloride by distillation under reduced pressure. Add dichloromethane (200 mL) to dissolve, add tert-butanol (12.38 mL, 129.48 mmol) and pyridine (9.1 mL, 146.64 mmol), add pyridine within 2 hours, and react at 25 °C for 3 h. Evaporate the solvent under reduced pressure, add 800 mL of a pre-cooled DMF / water mixed solvent (DMF and water are mixed in a volume ratio of 1:2), filter to collect the precipitate. Dry in vacuo to constant weight, re-suspend with dichloromethane (500 mL), collect the filtrate and evaporate the solvent under reduced pressure, re-suspend with petroleum ether (800 mL), collect the filtrate and evaporate under reduced pressure, add n-heptane (150 mL) for recrystallization, filter to collect the precipitate, and dry under reduced pressure to constant weight to obtain monotert-butyl docosanedioate, with a yield of 13.4 g and a yield of 29.1%; the purity detected by HPLC is 97.5%.

[0302] MS test of monotert-butyl docosanedioate ESI-MS m / z: 326.49 [M+H] + , which is consistent with the theoretical value.

[0303] Example 20

[0304] Synthesis of Monotert-butyl Docosanedioate-OSu

[0305]

[0306] Add monotert-butyl docosanedioate (10 g, 23.44 mmol), N-hydroxysuccinimide (3.8 g, 32.82 mmol) and 100 mL of DCM to a 250 mL single-necked flask, cool to -10 °C, add dicyclohexylcarbodiimide (6.8 g, 32.82 mmol) and continue to react for 2 hours, then return to room temperature and continue stirring for 6 hours. After the reaction is completed, filter to remove the precipitate, concentrate the filtrate by distillation under reduced pressure to dryness to obtain a crude solid, add 80 mL of isopropanol and 80 mL of n-hexane for recrystallization, filter, and wash the filter cake with 20 mL of n-hexane, and dry under reduced pressure to constant weight. Obtain 11.2 g of white solid monotert-butyl docosanedioate-OSu, with a yield of 91.2%, and the detected HPLC purity is 94.37%.

[0307] MS test of monotert-butyl docosanedioate-OSu: ESI-MS m / z: 523.89 [M+H] + , which is consistent with the theoretical value.

[0308] Example 21

[0309] Synthesis of tert-butyl ester - docosanedioyl - N6 - ((benzyloxy)carbonyl)-L-lysine tert-butyl ester

[0310]

[0311] Add tert-butyl docosanedioate-OSu (10.0 g, 19.1 mmol), N6 - ((benzyloxy)carbonyl)-L-lysine tert-butyl ester (7.7 g, 22.92 mmol) and 120 mL of THF into a 250 mL single-necked flask. Place it in an oil bath at 30 °C, add triethylamine (4.44 g, 43.93 mmol) and continue the reaction for 12 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L) and water to quench the reaction, extract, dry, and concentrate the filtrate under reduced pressure to dryness to obtain a crude solid. Add 90 mL of isopropanol and 30 mL of n-hexane for recrystallization, filter, and wash the filter cake with 30 mL of n-hexane, and dry under reduced pressure to constant weight. Obtain 12.5 g of white solid of tert-butyl ester - docosanedioyl - N6 - ((benzyloxy)carbonyl)-L-lysine tert-butyl ester, with a yield of 88.03% and a HPLC purity of 94.47% for testing.

[0312] MS test of tert-butyl ester - docosanedioyl - N6 - ((benzyloxy)carbonyl)-L-lysine tert-butyl ester: ESI-MS m / z: 745.61 [M + H] + , consistent with the theoretical value.

[0313] Example 22

[0314] Synthesis of tert-butyl ester - docosanedioyl - L-lys-tert-butyl ester

[0315]

[0316] Add tert-butyl ester - docosanedioyl - N6 - ((benzyloxy)carbonyl)-L-lysine tert-butyl ester (10 g, 13.42 mmol) and THF (100 mL) into a 250 mL single-necked reaction flask, add 12% Pd / C (1.2 g) and 0.1 mL of trifluoroacetic acid. After replacing the system with hydrogen three times, put on a hydrogen balloon and stir at 30 °C for 4 hours. After the reaction is completed, filter Pd / C and distill the filtrate to dryness under reduced pressure. Obtain 7.8 g of crude product. The yield is 95.1% and the HPLC purity for testing is 97.38%.

[0317] MS test of tert-butyl ester - docosanedioyl - L-lys-tert-butyl ester: ESI-MS m / z: 611.06 [M + H] +, in line with the theoretical value.

[0318] Example 23

[0319] Synthesis of tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl

[0320]

[0321] Add tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester (10 g, 16.38 mmol), succinic anhydride (2.45 g, 24.57 mmol) and 100 mL of acetonitrile into a 250 mL single-necked flask, place it in an oil bath at 30 °C, add triethylamine (3.5 g, 34.4 mmol) and continue the reaction for 10 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L with water) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 90 mL of isopropanol and 30 mL of n-hexane for recrystallization, filter, and wash the filter cake with 20 mL of n-hexane, then dry under reduced pressure to constant weight. 10.4 g of tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl is obtained, with a yield of 89.27% and the HPLC purity for inspection is 93.70%.

[0322] MS test of tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl: ESI-MS m / z: 711.59 [M+H] + , in line with the theoretical value.

[0323] Example 24

[0324] Synthesis of tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl - OSu

[0325]

[0326] Add tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl (10 g, 14.06 mmol), N-hydroxysuccinimide (2.1 g, 18.3 mmol) and 100 mL of DCM into a 250 mL single-necked flask, cool down to -10 °C, add dicyclohexylcarbodiimide (3.8 g, 18.3 mmol) and continue the reaction for 2 hours, then restore to room temperature and stir for another 3 hours. After the reaction is completed, filter to remove the precipitate, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a crude solid. Add 90 mL of ethanol for recrystallization, filter, and wash the filter cake with 10 mL of n-hexane, then dry under reduced pressure to constant weight. 9.6 g of white solid of tert-butyl ester - docosanedioyl - L-lys - tert-butyl ester - diacyl - OSu is obtained, with a yield of 84.5% and the HPLC purity for inspection is 98.89%.

[0327] tert-Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - OSu MS test: ESI - MS m / z: 808.78 [M + H] + , which is in line with the theoretical value.

[0328] Example 25

[0329] Synthesis of tert - Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - 2xOEG

[0330]

[0331] Add tert - Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - OSu (10 g, 12.4 mmol), 17 - amino - 10 - oxo - 3,6,12,15 - tetraoxo - 9 - azanonanoic acid (5.4 g, 17.36 mmol) and 100 mL of DMF into a 250 - mL single - necked flask. Place it in an oil bath at 30 °C, add triethylamine (2.6 g, 26.0 mmol) and continue the reaction for 8 hours. After the reaction is completed, add 2M HCl (197.3 g of 37% commercially available hydrochloric acid, made up to 1 L with water) and water to quench the reaction, extract, dry, concentrate the filtrate under reduced pressure by distillation to dryness to obtain a solid crude product. Add 100 mL of isopropanol and 35 mL of n - heptane for recrystallization, filter, and wash the filter cake with 15 mL of n - heptane, then dry under reduced pressure to constant weight. Obtain 8.9 g of tert - Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - 2xOEG, with a yield of 71.2% and the HPLC purity for submission is 94.71%.

[0332] tert - Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - 2xOEG MS test: ESI - MS m / z: 1001.85 [M + H] + , which is in line with the theoretical value.

[0333] Example 26

[0334] Synthesis of tert - Butyl ester - docosanedioyl - L - lys - tert - butyl ester - diacyl - 2xOEG - OSu

[0335]

[0336] tert-Butyl ester-docosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG (10 g, 10.0 mmol), N-hydroxysuccinimide (1.5 g, 13.0 mmol) and 100 mL of DCM were added to a 250 mL single-necked flask. The temperature was lowered to -10 °C, and dicyclohexylcarbodiimide (2.7 g, 13.0 mmol) was added and the reaction continued for 2 hours. The temperature was restored to room temperature and stirring continued for 3 hours. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated to dryness under reduced pressure distillation to obtain a crude solid. 80 mL of ethanol was added for recrystallization, followed by filtration, and the filter cake was rinsed with 10 mL of petroleum ether and dried under reduced pressure to constant weight. 8.7 g of white solid of tert-butyl ester-docosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu was obtained, with a yield of 79.23%, and the HPLC purity submitted for inspection was 95.59%.

[0337] MS test of tert-butyl ester-docosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu: ESI-MS m / z: 1098.81 [M+H] + , which is in line with the theoretical value.

[0338] Example 27

[0339] Synthesis of docosanedioyl-L-lys-diacyl-2xOEG-OSu

[0340]

[0341] tert-Butyl ester-docosanedioyl-L-lys-tert-butyl ester-diacyl-2xOEG-OSu (10 g, 9.1 mmol), dichloromethane (50 mL), and trifluoroacetic acid (50 mL) were added to a 250 mL single-necked reaction flask and stirred at 0 °C for 3 h. After the reaction was completed, the liquid in the system was distilled to dryness under reduced pressure to obtain a crude yellow liquid. Chlorobenzene (50 mL) was added to the crude product to dissolve it. It was placed on a rotary evaporator again, and after reduced pressure distillation, a yellow liquid was obtained again. 50 mL of chlorobenzene was added again and it was placed on a rotary evaporator for reduced pressure distillation again. A yellow liquid was obtained again. The above operation was repeated once more by adding a solvent and then performing reduced pressure distillation. Finally, 7.2 g of viscous yellow liquid of docosanedioyl-L-lys-diacyl-2xOEG-OSu was obtained, with a yield of 80.26%, and the HPLC purity submitted for inspection was 93.84%.

[0342] MS test of docosanedioyl-L-lys-diacyl-2xOEG-OSu: ESI-MS m / z: 986.59 [M+H] + , which is in line with the theoretical value.

[0343] Example 28

[0344] Preparation of A14E, B16E, B25H, B29K (Nε-eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG), DesB30 human insulin analogue

[0345] A14E, B16E, B25H, Des(B30) human insulin was prepared by a conventional method for preparing insulin analogues (for the specific method, see Tine Glendorf, Anders R Erica Nishimura, Ingrid Pettersson, Thomas Kjeldsen: Importance of the Solvent-Exposed Residues of the Insulin BChainα-Helix for Receptor Binding; Biochemistry 2008 47 4743-4751). 60 mg of A14E, B16E, B25H, Des(B30) human insulin was dissolved in 5 mL of water, and triethylamine (220 μL) was added to adjust the pH of the system to 12.10. According to the molar ratio of eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG-OSu: A14E, B16E, B25H, Des(B30) human insulin of 2.5:1, 40.6 mg of eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG-OSu was weighed in 5 mL of NMP (N-methylpyrrolidone), and then added to the insulin solution. After 15 minutes, the reaction was quenched by adding a mixed solution of 20% ethanolamine and 10% citric acid. Finally, 2M HCl was added to adjust the pH of the system to about 7.80. HPLC analysis showed that 29% of the acylated product was formed. After purification, an insulin analogue with a purity higher than 99.37% was obtained.

[0346] MS confirmation of A14E, B16E, B25H, B29K (Nε-eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG), DesB30 human insulin analogue: ESI-MS m / z: 6372.89 [M+H] + , which is in agreement with the theoretical value.

[0347] Example 29

[0348] Preparation of B29K (Nε-eicosanedicarbonyl-L-lys-dicarbonyl-2xOEG), DesB30 human insulin analogue

[0349] Des(B30) human insulin was prepared according to Example 1 of Patent CN1032900083B. 60 mg of Des(B30) human insulin was dissolved in 5 mL of water, and triethylamine (220 μL) was added to adjust the pH of the system to 12.10. 40.6 mg of eicosanedioyl-L-lys-diacyl-2xOEG-OSu was weighed according to the molar ratio of eicosanedioyl-L-lys-diacyl-2xOEG-OSu:Des(B30) human insulin of 2.5:1 in 5 mL of NMP, and then added to the insulin solution. After 15 minutes, the reaction was quenched by adding a mixed solution of 20% ethanolamine and 10% citric acid. Finally, 2M HCl was added to adjust the pH of the system to about 7.80. HPLC analysis showed that 33% of the acylated product was formed, and an insulin analogue with a purity higher than 98.60% could be obtained after purification.

[0350] Preparation of B29K(Nε-eicosanedioyl-L-lys-diacyl-2xOEG), DesB30 human insulin analogue MS confirmation: ESI-MS m / z: 6550.11 [M+H] + , which is in line with the theoretical value.

[0351] Example 30

[0352] Preparation of A14E, B16H, B25H, B29K(Nε-eicosanedioyl-L-lys-diacyl-2xOEG), DesB30 human insulin analogue

[0353] A14E, B16H, B25H, Des(B30) human insulin was prepared by a conventional method for preparing insulin analogues (for the specific method, see Tine Glendorf, Anders R Erica Nishimura, Ingrid Pettersson, Thomas Kjeldsen: Importance of the Solvent-Exposed Residues of the Insulin BChainα-Helix for Receptor Binding; Biochemistry 2008 47 4743-4751), Dissolve 60 mg of A14E, B16H, B25H, Des(B30) human insulin in 5 mL of water, and add triethylamine (220 μL) to adjust the pH of the system to 12.10. Weigh 40.6 mg of dicosanoyl-L-lysyl-diacyl-2xOEG-OSu according to the molar ratio of dicosanoyl-L-lysyl-diacyl-2xOEG-OSu: A14E, B16H, B25H, Des(B30) human insulin of 2.5:1 in 5 mL of NMP, and then add it to the insulin solution. After 15 minutes, quench the reaction by adding a mixed solution of 20% ethanolamine and 10% citric acid. Finally, add 2M HCl to adjust the pH of the system to about 7.80. HPLC analysis showed that 36% of the acylated product was formed, and an insulin analogue with a purity higher than 98.89% could be obtained after purification.

[0354] MS confirmation of A14E, B16H, B25H, B29K (Nε-dicosanoyl-L-lysyl-diacyl-2xOEG), DesB30 human insulin analogue: ESI-MS m / z: 6480.10 [M+H] + , which is consistent with the theoretical value.

[0355] Example 31

[0356] N-ε 26 -[Dicosanoyl-L-lysyl-diacyl-2xOEG], Preparation of [Gly8, Arg34]GLP-1(7-37) peptide

[0357] Prepare [Gly8, Arg34]GLP-1(7-37) peptide by a general protein recombinant expression method, such as the method described in reference patent CN111018964A. Take [Gly8, Arg34]GLP-1(7-37) peptide (2 g, 0.59 mmol) and dissolve it in 100M Na 2 HPO 4In an aqueous solution (65 mL), add NMP (35 mL), and adjust the pH of the system to between 10.50 and 12.00 with triethylamine. Dissolve docosanedioyl-L-lys-diacyl-2xOEG-OSu (0.68 g, 0.71 mmol) in a mixed solution of NMP / DMF (20 mL, volume ratio 1:1) in advance, and quickly add it to the reaction system to maintain the pH between 10.50 and 12.00. After stirring the reaction at 20 °C for 60 minutes, add hydrochloric acid solution to the system to adjust the pH to 7.8. HPLC analysis shows that 37% of the acylated product is formed. After purification, the title compound with a purity of 98.57% can be obtained.

[0358] N-ε 26 -[Docosanedioyl-L-lys-diacyl-2xOEG],[Gly8,Arg34]GLP-1(7-37) peptide MS confirmation: ESI-MS m / z: 4222.35 [M+H] + , which is consistent with the theoretical value.

[0359] Example 32

[0360] N-ε 26 - [Docosanedioyl-L-lys-diacyl-2xOEG],[Aib8,Arg34]GLP-1(7-37) peptide preparation

[0361] [Aib8,Arg34]GLP-1(7-37) peptide was prepared with reference to Example 29 of Patent CN105154498A. Dissolve [Aib8,Arg34]GLP-1(7-37) peptide (0.5 g, 0.15 mmol) in 100 mM Na 2 HPO 4 In an aqueous solution (15 mL), add NMP (15 mL), and adjust the pH of the system to between 10.50 and 12.00 with triethylamine. Dissolve docosanedioyl-L-lys-diacyl-2xOEG-OSu (0.19 g, 0.20 mmol) in a mixed solution of NMP / DMF (8 mL, volume ratio 1:1) in advance, and quickly add it to the reaction system to maintain the pH between 10.50 and 12.00. After stirring the reaction at 20 °C for 60 minutes, add 2M hydrochloric acid solution to the system to adjust the pH to 7.5. HPLC analysis shows that 47% of the acylated product is formed. After purification, the title compound with a purity of 97.65% can be obtained.

[0362] N-ε 26-[Docosanoyl-L-lysyl-diyl-2xOEG],[Aib8,Arg34]GLP-1(7-37) peptide MS confirmation: ESI-MS m / z: 4240.82 [M+H] + , which is consistent with the theoretical value.

[0363] Example 33

[0364] N-ε 20 -[Docosanoyl-L-lysyl-diyl-2xOEG],[Aib2, Aib13, Ser(NH 2 )39]GLP-1 / GIP(1-39) peptide or preparation of tirzepatide

[0365] [Aib2,Aib13,Ser(NH 2 )39]GLP-1 / GIP(1-39) tirzepatide fully protected was prepared with reference to Patent WO2020 / 159949A1 to obtain (Boc-(tBu)Y-{Aib}-(tBu)E-G-(tBu)T-F-(tBu)T-(tBu)S-(tBu)D-(tBu)Y-(tBu)S-I-{Aib}-L-(tBu)D-(Boc)K-I-A-(Trt)Q-K-A-F-V-(Trt)Q-(Boc)W-L-I-A-G-G-P-(tBu)S-(tBu)S-G-A-P-P-P-(tBu)S-NH 2 ).

[0366] Boc-(tBu)Y-{Aib}-(tBu)E-G-(tBu)T-F-(tBu)T-(tBu)S-(tBu)D-(tBu)Y-(tBu)S-I-{Aib}-L-(tBu)D-(Boc)K-I-A-(Trt)Q-K-A-F-V-(Trt)Q-(Boc)W-L-I-A-G-G-P-(tBu)S-(tBu)S-G-A-P-P-P-(tBu)S-NH 2The polypeptide with side chain protecting group (33.2 mg, 0.006 mmol) was dissolved in dichloromethane (4 mL) before adding diisopropylethylamine (3.1 mg, 0.024 mmol). Eicosanedioyl-L-lys-diyl-2xOEG-OSu (14.4 mg, 0.015 mmol) was pre-dissolved in DMF (1 mL) and quickly added to the reaction system. The reaction was stirred at 20 °C for 120 minutes, and then the reaction solution was concentrated under vacuum to obtain an oily substance. Then, 200 μL of a solution containing trifluoroacetic acid / diisopropylsilane (volume ratio of trifluoroacetic acid, diisopropylsilane and water is 95:2.5:2.5) was added, and the reaction was continued to stir for 18 hours. After the reaction was completed, it was distilled under reduced pressure at 25 °C to obtain an oily substance, and then it was dissolved with 1 mL of DMF and 5 mL of Na 2 HPO 4 (100 mM) mixed solvent. HPLC analysis showed that 29% of the target product was formed. After purification, the title compound with a purity of 95.05% was obtained.

[0367] N-ε 20 -[Eicosanedioyl-L-lys-diyl-2xOEG],[Aib2,Aib13,Ser(NH 2 )39]GLP-1 / GIP(1-39) peptide MS confirmation: ESI-MS m / z: 4911.60 [M+H] + , which is in line with the theoretical value.

[0368] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0369] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing long-chain fatty diacid mono-tert-butyl ester, characterized in that: The following steps are involved: S1: subjecting a long fatty chain diacid to an acyl chlorination reaction to obtain a long fatty chain diacid chloride; S2: contacting the long fatty chain diacyl chloride with tert-butyl alcohol and an organic base reagent a to obtain long fatty chain diacyl monotert-butyl ester; Wherein, the long fatty chain diacid has a structure shown in formula (A); X is an integer of 6-32.

2. The method according to claim 1, characterized in that Step S1 further comprises subjecting the long fatty chain diacid to an acyl chlorination reaction with an acyl chlorination agent to obtain a long fatty chain diacid chloride; Optionally, the molar amount of the acyl chloride reagent is equivalent to 3 to 16 times the molar amount of the long fatty chain diacid; Optionally, the chlorination agent comprises at least one selected from thionyl chloride, phosphorus pentachloride, phosphorus trichloride, phosphorus oxychloride, and oxalyl chloride; Optionally, the acyl chlorination reaction is carried out at a temperature of 70°C to 120°C for 3 to 24 hours or at 15°C to 30°C in the presence of a catalyst; Optionally, the catalyst is N,N-dimethylformamide.

3. The method according to claim 1, characterized in that The organic base reagent a in step S2 includes at least one selected from ethylenediamine, triethylamine, diisopropylethylamine, pyridine, and piperidine; preferably pyridine; Optionally, the molar amount of the organic base reagent a is equivalent to 0.6 to 1.5 times the molar amount of the long fatty chain diacid; preferably 1.0 to 1.2 times; more preferably 1.04 to 1.05 times; Optionally, the temperature at which the long fatty chain diacyl chloride is contacted with tert-butyl alcohol and the organic base reagent a in step S2 is 20° C. to 30° C., preferably 20° C. to 25° C.; Optionally, the time for the long fatty chain diacyl chloride to contact with tert-butyl alcohol and organic base reagent a in step S2 is 2 to 16 hours; preferably 2 to 4 hours; more preferably 3 hours; Optionally, step S2 further comprises contacting the long fatty chain diacyl chloride with tert-butyl alcohol and an organic base reagent a to obtain a mixed solution, dissolving the mixed solution in a first solvent, and collecting a precipitate; The first solvent comprises at least one selected from N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran; preferably N,N-dimethylformamide; Optionally, the temperature of the first solvent is 0 to 4°C; Optionally, the volume fraction of the N,N-dimethylformamide, acetonitrile, acetone, and tetrahydrofuran in the first solvent is 16.7% to 50%; preferably 25% to 33.3%; more preferably 33.3%; Optionally, the volume mass ratio of the first solvent to the long fatty chain diacid is (10-50 mL): 1 g; preferably (20-30 mL): 1 g; more preferably 30 mL: 1 g; Optionally, step S2 further comprises washing and recrystallizing the precipitate; Optionally, the washing treatment is carried out in at least one second solvent selected from the group consisting of dichloromethane, ethyl acetate, petroleum ether, and n-heptane; preferably petroleum ether; Optionally, the volume mass ratio of the second solvent to the long fatty chain diacid is (10-50 mL): 1 g; preferably (10-20 mL): 1 g; Optionally, the recrystallization treatment is carried out in a third solvent; the third solvent is petroleum ether or n-heptane; preferably n-heptane; Optionally, the volume mass ratio of the third solvent to the long fatty chain diacid is 4 mL:1 g.

4. A method for preparing a long fatty chain diacid derivative, characterized in that: The following steps are involved: (1) preparing a long fatty chain diacid according to the method described in any one of claims 1 to 3 to obtain long fatty chain diacid mono-tert-butyl ester, wherein the long fatty chain diacid has a structure shown in formula (A); (2) subjecting the long fatty chain diacid mono-tert-butyl ester to an esterification reaction with N-hydroxysuccinimide to obtain long fatty chain diacid succinimide tert-butyl ester; (3) subjecting the long fatty chain diacid succinimide tert-butyl ester to a nucleophilic addition amidation reaction with the compound represented by formula (B) to obtain the compound represented by formula (C); (4) subjecting the compound represented by formula (C) to a hydrogenation reaction to obtain a compound represented by formula (D); (5) subjecting the compound represented by formula (D) to a nucleophilic addition amidation reaction with the compound represented by formula (E) to obtain a compound represented by formula (F); (6) subjecting the compound represented by formula (F) to an esterification reaction with N-hydroxysuccinimide to obtain a compound represented by formula (G); (7) subjecting the compound represented by formula (G) to a nucleophilic addition amidation reaction with the compound represented by formula (H) to obtain the compound represented by formula (I); (8) subjecting the compound represented by formula (I) to an esterification reaction with N-hydroxysuccinimide to obtain a compound represented by formula (J); (9) subjecting the compound represented by formula (J) to a deprotection reaction to obtain a compound represented by formula (K); Wherein, X is an integer of 6 to 32; Y is an integer of 2 to 8; and Z is an integer of 1 to 12.

5. The method according to claim 4, characterized in that The esterification reaction in step (2) is carried out in at least one fourth solvent selected from N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, acetonitrile and water at -10°C to 20°C for 2 hours and at 15°C to 40°C for 3 to 24 hours; Optionally, the volume mass ratio of the fourth solvent to the long fatty chain diacid mono-tert-butyl ester is (5-20 mL): 1 g; Optionally, step (2) further comprises contacting the long fatty chain diacid mono-tert-butyl ester with dicyclohexylcarbodiimide; Optionally, the molar amount of the N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain diacid mono-tert-butyl ester; Optionally, the molar amount of the dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain diacid mono-tert-butyl ester; Optionally, step (2) further comprises filtering, crystallizing, and washing the system after the esterification reaction; Optionally, the washing treatment is carried out in at least one fifth solvent selected from the group consisting of methanol, ethanol, isopropanol, tert-butanol, n-hexane, n-octane, and cyclohexane; Optionally, the volume mass ratio of the fifth solvent to the long fatty chain diacid mono-tert-butyl ester is (1-3 mL): 1 g.

6. The method according to claim 4, characterized in that The nucleophilic addition amidation reaction in step (3) is carried out in at least one sixth solvent selected from chloroform, N,N-dimethylformamide, dimethylacetamide, dimethyl sulfoxide, dichloromethane, and water at 20° C. to 40° C.; Optionally, the volume mass ratio of the sixth solvent to the long fatty chain diacid succinimide tert-butyl ester is (5-20 mL): 1 g; Optionally, the molar amount of the compound represented by formula (B) is equivalent to 1.0 to 1.8 times the molar amount of the long fatty chain diacid succinimide tert-butyl ester; Optionally, step (3) further comprises contacting the long fatty chain diacid succinimide tert-butyl ester with at least one organic base reagent b selected from 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, piperidine, pyridine, ethanolamine, ethylenediamine, and triethylamine; Optionally, the molar amount of the organic base reagent b is equivalent to 1.5 to 3.0 times the molar amount of the long fatty chain diacid succinimide tert-butyl ester.

7. The method according to claim 4, characterized in that The hydrogenation reaction in step (4) is carried out in at least one seventh solvent selected from acetonitrile, methanol, acetone, tetrahydrofuran, ethanol, dichloromethane and chloroform, reacting with hydrogen at 20° C. to 35° C. for 1 to 3 hours; Optionally, the volume mass ratio of the seventh solvent to the compound represented by formula (C) is (5-20 mL): 1 g; Optionally, the hydrogenation reaction is carried out in the presence of a catalyst; Optionally, the catalyst is Pd / C; Optionally, the mass ratio of the catalyst to the compound represented by formula (C) is (0.05-0.15):

100.

8. The method according to claim 4, characterized in that The nucleophilic addition amidation reaction in step (5) is carried out in at least one eighth solvent selected from the group consisting of dichloromethane, ethyl acetate, acetone, methanol, and acetonitrile at 25° C. to 40° C.; Optionally, the volume mass ratio of the eighth solvent to the compound represented by formula (D) is (5-20 mL): 1 g; Optionally, the molar amount of the compound represented by formula (E) is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (D); Optionally, step (5) further comprises contacting the compound represented by formula (D) with at least one organic base reagent c selected from the group consisting of sodium hydroxide, potassium tert-butoxide, triethylamine, 4-dimethylaminopyridine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene; Optionally, the molar amount of the organic base reagent c is equivalent to 2.0 to 3.0 times the molar amount of the compound represented by formula (D); Optionally, step (5) further comprises crystallizing and washing the system after the nucleophilic addition amidation reaction; Optionally, the washing treatment is carried out in at least one ninth solvent selected from the group consisting of isopropanol, n-heptane, ethanol, methanol, cyclohexane, ethyl acetate, acetonitrile, n-hexane, petroleum ether, and diethyl ether; Optionally, the volume mass ratio of the ninth solvent to the compound represented by the formula (D) is (3-20 mL): 1 g.

9. The method according to claim 4, characterized in that The esterification reaction in step (6) is carried out in at least one of the tenth solvents selected from the group consisting of dichloromethane, methyl tert-butyl ether, 1,4-dioxane, tetrahydrofuran, water, toluene and chlorobenzene, at -10°C to 10°C for 2 hours and at 15°C to 40°C for 3 to 24 hours; Optionally, the volume mass ratio of the tenth solvent to the compound represented by the formula (F) is (5-20 mL): 1 g; Optionally, the molar amount of the N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (F); Optionally, step (6) further comprises contacting the compound represented by formula (F) with dicyclohexylcarbodiimide; Optionally, the molar amount of the dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (F); Optionally, step (6) further comprises filtering, crystallizing, and washing the system after the esterification reaction; Optionally, the washing treatment is carried out in at least one eleventh solvent selected from the group consisting of diethyl ether, ethanol, ethyl acetate, isopropyl acetate, petroleum ether, isopropanol, n-heptane, and n-octane; Optionally, the volume mass ratio of the eleventh solvent to the compound represented by formula (F) is (3-20 mL): 1 g.

10. The method according to claim 4, characterized in that The nucleophilic addition amidation reaction in step (7) is carried out in at least one twelfth solvent selected from the group consisting of dichloromethane, ethanol, acetonitrile, N,N-dimethylformamide, and acetone at a temperature of 25° C. to 40° C.; Optionally, the volume mass ratio of the twelfth solvent to the compound represented by the formula (G) is (5-20 mL): 1 g; Optionally, step (7) further comprises contacting the compound represented by formula (G) with at least one organic base reagent d selected from triethylamine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, tetramethylhexanediamine, and piperidine; Optionally, the molar amount of the organic base reagent d is equivalent to 2.0 to 3.0 times the molar amount of the compound represented by the formula (G); Optionally, step (7) further comprises crystallizing and washing the system after the nucleophilic addition amidation reaction; Optionally, the washing treatment is carried out in at least one thirteenth solvent selected from the group consisting of petroleum ether, n-hexane, cyclohexane, n-heptane, ethyl acetate, isopropanol, and ethanol; Optionally, the volume mass ratio of the thirteenth solvent to the compound represented by the formula (G) is (3-20 mL): 1 g; Optionally, the molar amount of the compound represented by formula (H) is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (G).

11. The method according to claim 4, characterized in that The esterification reaction in step (8) is carried out in at least one of the fourteenth solvents selected from the group consisting of dichloromethane, tetrahydrofuran, acetonitrile, p-xylene, o-xylene and ether, at -10°C to 10°C for 2 hours and at 15°C to 40°C for 3 to 24 hours; Optionally, the volume mass ratio of the fourteenth solvent to the compound represented by the formula (I) is (5-20 mL): 1 g; Optionally, the molar amount of the N-hydroxysuccinimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (I); Optionally, step (8) further comprises contacting the compound represented by formula (I) with dicyclohexylcarbodiimide; Optionally, the molar amount of the dicyclohexylcarbodiimide is equivalent to 1.0 to 1.8 times the molar amount of the compound represented by formula (I); Optionally, step (8) further comprises filtering, crystallizing, and washing the system after the esterification reaction; Optionally, the washing treatment is carried out in at least one fifteenth solvent selected from the group consisting of isopropanol, ethanol, tert-butanol, n-heptane, n-hexane, and n-octane; Optionally, the volume mass ratio of the fifteenth solvent to the compound represented by formula (I) is (3-20 mL): 1 g.

12. The method according to claim 4, characterized in that The deprotection reaction in step (9) is carried out in at least one of the sixteenth solvents selected from ethyl acetate, isopropanol, ethanol, chloroform, dichloromethane and N,N-dimethylformamide, in contact with trifluoroacetic acid at 0 to 20° C.; Optionally, the volume mass ratio of the sixteenth solvent to the compound represented by the formula (J) is (5-20 mL): 1 g; Optionally, the volume mass ratio of the trifluoroacetic acid to the compound represented by the formula (J) is (5-10 mL): 1 g; Optionally, step (9) further comprises dissolving the system after the deprotection reaction in at least one seventeenth solvent selected from the group consisting of p-xylene, chlorobenzene, toluene, tetrahydrofuran and diethyl ether and performing a reduced pressure distillation operation; Optionally, the volume mass ratio of the seventeenth solvent to the compound represented by formula (J) is (5-20 mL): 1 g.

13. The method according to any one of claims 4 to 12, characterized in that: The Y is 2, 4, 6 or 8; preferably 4; Optionally, the structure of the compound represented by formula (B) is Optionally, the structure of the compound represented by formula (C) is Optionally, the structure of the compound represented by formula (D) is Optionally, Z is an integer of 2 to 6; preferably 2; Optionally, the structure of the compound represented by formula (F) is Optionally, the structure of the compound represented by formula (G) is Optionally, the structure of the compound represented by formula (I) is Optionally, the structure of the compound represented by formula (J) is 14. Use of the method according to any one of claims 4 to 13 in the preparation of insulin analogs, GLP-1 analogs, GLP-1 / GIP analogs or GLP-1 / GIP / GCG analogs.

15. A method for preparing a blood sugar-lowering protein drug, characterized in that: The method comprises: modifying the protein with a long fatty chain diacid derivative to obtain the blood sugar lowering protein drug; wherein the long fatty chain diacid derivative is obtained by the method according to any one of claims 4 to 13; Optionally, the blood sugar lowering protein drug includes at least one selected from insulin analogs, GLP-1 analogs, GLP-1 / GIP analogs, and GLP-1 / GIP / GCG analogs; Optionally, the protein is Icodec insulin, liraglutide, semaglutide or telpotide; Optionally, the modification is a fatty side chain modification.

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